Hi! Cloning a double blunt piece is always troublesome. Dephosphorylation
may help but it is pain to do. In terms of the second ligation, I think it
may help if you can use the same brand ligase and buffer since they are
optamized accordingly, and you may want to check out the unit definition
with different brand enzymes. In addition, optamizing the ration of insert
vs vector on the basis of molarity not weight (moles, not ug). These are
the suggestion I can provided based on the info in your mail.